KR20120077691A - A cordycepin producing method using unhatched egg - Google Patents
A cordycepin producing method using unhatched egg Download PDFInfo
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- KR20120077691A KR20120077691A KR1020100139750A KR20100139750A KR20120077691A KR 20120077691 A KR20120077691 A KR 20120077691A KR 1020100139750 A KR1020100139750 A KR 1020100139750A KR 20100139750 A KR20100139750 A KR 20100139750A KR 20120077691 A KR20120077691 A KR 20120077691A
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- cordyceps
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- cordyceps sinensis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
Abstract
The present invention relates to a method for producing cordycepin by inoculating and incubating pre-cultivated Cordyceps fungi while adding lactic acid to the unhatched ovulation generated in the hatchery, dissolving sterilization and egg shells, and then aeration into a fermentor. Representative cordycepin in the active ingredient of Cordyceps sinensis was obtained in an amount of about 356 mg / L in a culture solution cultured for 20 days by the present invention.
Description
The present invention relates to a method for producing cordycepin by inoculating and fermenting Cordyceps fungi to fermented unhatched embryos, and more specifically, to dissolve eggshells of unhatched eggs with lactic acid and inoculating Cordyceps fungi precultured in a fermenter and controlling aeration. By fermentation to produce a large amount of cordycepin (cordycepin), an important bioactive substance of Cordyceps sinensis.
Cordyceps sinensis is a type of mushroom belonging to the genus Cordyceps sinensis of Cordyceps sinensis, about 800 species have been reported around the world, and about 70 species have been reported in Korea. While most mushrooms grow nutrients from plants and soil to mycelium and convert to reproductive stages due to seasonal causes, cordyceps invades animal seeds, spiders, arthropods, fungi or seeds of higher plants. This is characterized by the growth of mycelia and the formation of fruiting bodies after the growth of mycelium. The name of Cordyceps sinensis is 한 夏 草 in Chinese characters, and it is characteristic of the life history of Cordyceps sinensis, meaning that it was formed in the winter, and fruiting bodies like grasses in summer.
Cordyceps sinensis is known as a rare rare old age potion in the Silver Age, and jade stones such as Cordyceps were found in the tombs of kings and emperors from 1,000 BC to 200 BC. Cordyceps, which were taken by Qin Shi Huang and Poppies, was recorded in the herbaceous life of Qing Dynasty Oui Lak and distilled herbaceous flowers of 1,082 in Japan. In modern times, Cordyceps became widely known both domestically and in the world. In the 1980s, Deng Xiaoping was used to maintain health, and many records came from Chinese athletes taking Cordyceps at the August 1993 World Athletics Championships. It was a chance.
In China, bat moth Cordyceps sinensis and chrysalis are popular in alpine areas such as Tibet, Qinghai and Sichuan. The production of Cordyceps, which is based on bat moths, is produced by collecting people from 3,000 to 6,000 meters above sea level in Tibet and Nepal's Sichuan, Qinghai and Yunnan provinces, and in demanding growth conditions, climate change and overfishing. It is gradually decreasing due to the cause, and is becoming more rare and increasing in price. Recently, China has been cultivating Cordyceps sinensis with modern technology.
In Korea, as a result of the research on the sleep insects, Paecillomyces japonica production technology has been widely distributed, followed by cultivation and use of chrysalis insects (Cordyceps militaris).
Cordyceps are highly diverse and are found in cicadas, stink bugs, scarabs, ants, dragonflies, moths, butterflies, dust beetles, bees, spiders, and flies. According to research by Sung Jae Mo, various kinds of Cordyceps sinensis have been found in Korea.
Cordyceps sinensis is produced by inoculating silkworms with the inoculation of Cordyceps sinensis, making silkworms form cocoons, and growing Cordyceps sinensis to form fruiting bodies. Inoculation of Cordyceps sinensis to the nutritional components of the plant has been carried out. However, these methods take time and effort to raise and manage silkworms using silkworms and pupa, and the success rate was not high. In addition, as the diet improves, foods such as pupa and silkworms are often avoided by some people and are generally limited.
Cordyceps production in Korea is mainly produced using the pupa of silkworms and silkworms. It takes about 25 days for the silkworms to grow to be silkworms of the 5th age, and it takes about 60 days to inoculate the cordyceps fungi and become cordyceps. This takes
Silkworms eat only mulberry leaves. In the mulberry leaves and the growing environment that silkworms eat, pesticides and harmful chemicals can cause the silkworms to die quickly. Therefore, in order to raise silkworms, they must be deep in the mountains or in clean areas. Silkworm breeding is very demanding and special care should be taken when the silkworm eggs wake up and young silkworms grow. For young silkworms, the mulberry leaves are finely chopped and the amount of mulberry leaves eaten by the rapid growth of silkworms is not easy to harvest, carry and feed the mulberry in the field, and can produce twice in spring and autumn.
Cordyceps sinensis is naturally expensive (43 million won / kg of Chinese bat moth), as well as artificially cultivated in Korea, which is very expensive (150g 120,000 won of Jeju Militaris cordyceps).
Meanwhile, eggs consume 11kg per person in 2008, and domestic daily circulation amounts to 25 million pieces, and it is easy to buy them on the market. One egg is very cheap (180 ~ 700 won / dog) even though it is a complete nutritious food containing all the nutrients evenly enough to be one chick.
If the pharmacological component of Cordyceps sinensis is added to these eggs, it will be very useful for maintaining the health of people.
In general, the pharmacological components of Cordyceps sinensis are known as cordycepin, cordycepic polysaccharides, cordycepic acid, mannitol, amino acids, and vitamin precursors. The main pharmacological functions of Cordyceps sinensis are nourishing tonic function, immune enhancing function, natural hemorrhage, anticancer function, drug addiction detoxification function, exercise ability improvement function, inflammation suppression function, arteriosclerosis improvement function, diabetes improvement function, respiratory improvement function, kidney It is known to have an effect of improving function, enhancing energy and suppressing blood pressure increase in hypertension.
According to the data released by the Agricultural Monitoring Center in March 2010, the production of broiler eggs is 66,363.6 million per year, and the hatching rate is estimated at 70%, and the production of broiler eggs is 1.152.25 million per year, 30% of which is about 30 million hatched by-products. Disturbance occurs. If it is calculated at 60g per piece, the annual occurrence of disturbance is 200,000205 tons. It was illegally collected and used as a raw material for bakery, resulting in social controversy. Disposal is not possible to use for food, it is disposed of on the farm is used as a compost paying waste disposal costs. However, since egg content is about 74% except eggshell, it is not welcomed as a waste.
The inventors of the present invention show that the egg contains no eggshell, and the chemical composition is 72.5% of moisture, 13.3% of crude protein, 11.6% of lipids, 1.5% of sugars, and 1.1% of ash. 22.2% protein, 13.3% lipid, 1.9% carbohydrate, 13% ash, it is considered that eggs are suitable for cultivation of cordyceps. If the eggs are collected as waste, the eggs are collected and developed into cordycepin material with excellent functionality. It is highly desirable to reduce waste and develop it into a very high value added product.
In the production of Cordyceps sinensis using eggs, which are not ovulation, the method of cultivating Snow Cordyceps sinensis using eggs produced by dividing the eggs into the beaker, cultivating Snow Cordyceps and growing fruit bodies has been registered (Patent No. 10-0411703). .
Eggs form whole eggs in the uterus of the chicken and spawn into the total excretory cavity. Eggs are contaminated while the eggs pass through the total excretory cavity. At this time, the bacteria that contaminate the total excretory cavity of the chicken are micrococci and entero. Enterococci , Coli-aerogenes , and the like. Microorganism which is detected in the egg shell is priced at about 10 5, and its type is a micro Rhodococcus (Micrococcus), Staphylococcus (Staphylococcus), bakteo (Arthrobacter), Bacillus (Bacillus), Sar or when (Sarcina), Pseudomonas (Pseudomonas) in art , Achromobacter , Escherichia , Aerobacter and the like.
In order to inoculate Cordyceps fungus into eggs, it is necessary to divide egg shells and to use egg shells to separate egg shells from egg shells. The egg contents are highly contaminated in this process. As a way to reduce contamination, the surface of the egg is sterilized with ozone water, sodium hypochlorite and 70% ethanol, and the harlan process is performed in a clean room, but the installation cost is still perfect. Could not prevent pollution.
In addition, when the eggs are heat-treated, egg proteins are heat-changed to become gels, which makes it impossible to agitate and aeration. Therefore, normal steam or heat sterilization is not suitable for egg culture. In addition, when the egg is cultured through aeration, a large amount of foam is generated due to the components contained in the egg white of the egg, which causes the foam to overflow to the outside during fermentation, so that the contents are all leaked out and cause contamination.
Therefore, an object of the present invention is to develop an efficient sterilization method which is a problem in using the embryonic eggs as a host of cordyceps, and to provide a method for producing a large amount of cordycepin derived from expensive cordyceps using the embryo.
The present inventors studied a method of utilizing the unhatched ovulation which is discarded without any use, in the culture of Cordyceps sinensis. At this time, the long-term research was conducted to efficiently sterilize the problematic eggs and cultivate Cordyceps while maintaining the high calcium content of the eggs, and sterilize eggshells using lactic acid, as well as separate eggshells. The method of producing cordycepin was completed by culturing Cordyceps fungi without solidifying the bacteria. As a result of this study, cordycepin derived from Cordyceps sinensis, which is known for the treatment of diabetes, prevention of vascular restenosis, antiviral, obesity, intestinal action, and inflammatory treatment, can be efficiently cultured and produced in the lungs.
The present invention
a) dissolving and sterilizing the eggshell by adding lactic acid to the unriched egg of algae;
b) inoculating the pre-cultured Cordyceps sinensis solution in the sterilized ovulation obtained in step a) and culturing with aeration of 0.1 ~ 0.5vvm; and relates to a cordycepin production method using undigested ovulation.
The method of adding lactic acid to unhatched ovulation is very effective as a sterilization method and is very useful because calcium components of eggshells can be contained in the culture medium without breaking and removing eggshells.
In addition, in the Cordyceps cultivation step, the ventilation amount should be carefully controlled, and the preferable ventilation amount is 0.1 to 0.5 vvm. Excessive aeration is undesirable because it causes excessive foaming, which causes the culture liquid to overflow or lead to contamination, and when the amount of ventilation is too small, smooth culture does not occur.
The present invention also relates to a method for producing cordycepin, wherein the step of purifying cordycepin after step b) is added.
In addition, the present invention is characterized in that the culture step of b) is carried out for 5 to 30 days. Incubation in less than 5 days does not produce enough cordycepin, which is uneconomic for more than 30 days.
In addition, the present invention is characterized in that the cordyceps is Cordyceps Militaris. As a result of the experiment of the present inventors, cordyceps millitaris was more suitable for cultivation in ovulation than other kinds of cordyceps, and a large amount of cordycepin could be obtained.
According to the method of the present invention, it is possible to sterilize Cordyceps, which is difficult to culture, by a simple method, and can significantly reduce the production cost of Cordyceps. Therefore, by using the method of the present invention can be produced inexpensively cordyceps and cordycepin showing a variety of physiological activity.
In addition, according to the method of the present invention, cordycepin, which is a useful ingredient included in Cordyceps sinensis, can also be produced in large quantities, and thus it can be used as a material for medicine, animal medicine, feed, cosmetics, and the like.
1 is a chromatogram showing the result of diluting 100-fold dilution after pretreatment of incubated Cordyceps sinensis for 20 days.
Hereinafter, the configuration of the present invention will be described in detail with reference to specific embodiments. However, it will be apparent to those skilled in the art that the scope of the present invention is not limited by the description of the embodiments.
Example 1: Cordyceps sinensis preculture
Cordyceps militaris was used to culture Cordyceps fungi. 200 ml of distilled water was added to a 500 ml Erlenmeyer flask, and 2.4 g of PDB (Photato Dextrose Broth, Difco) and 1.4 g of agar were stirred for 10 minutes, then capped with a face and sterilized at 121 ° C. for 15 minutes in an autoclave. . About 20 ml of sterilized PDA medium in a cleanbench was dispensed into a petri dish of 90 mm in diameter and completely cooled.
Mycelium masses of Cordyceps fungi precultured using a corkborough of 10 mm in diameter were isolated, and then inoculated on the surface of the PDA medium and incubated at 25 ° C. for 20 days. After 20 days of culture, the hyphae showed 76 mm growth from the center. 100 ml of PDB medium was placed in a 500 ml Erlenmeyer flask, followed by sterilization at 121 ° C. for 15 minutes. The mycelium cultured in the PDA Petri dish was separated into three cultured masses by Kolkborough and inoculated into the medium in a sterilized Erlenmeyer flask, followed by shaking culture at 25 ° C. for 20 days. After shaking culture for 20 days, it was confirmed that mycelium formed pellets and a large amount of mycelium was grown.Then, the culture medium was homogeneously pulverized for 2 minutes at 20,000rpm using a homogenizer in a cleanbench and used as an inoculum. It was. The container and cutter used at this time were prepared by sterilizing at 121 ° C in advance.
Example 2: Cordyceps Sinensis Culture in Ovulation
40 unhatched ovules from the hatchery are placed in a 5-liter incubator and 90% lactic acid is added and soaked at 60 ° C. for 2 hours to melt the egg shells so that the eggs do not solidify. 100 ml of Cordyceps militaris precultured incubated in an Erlenmeyer flask was inoculated. After inoculation, the aeration rate was adjusted to 0.2-0.5vvm and incubated at 25 ° C. for 20 days while stirring.
Example 3: Analysis
After incubation, in order to determine the cordycepin content in the culture solution, 60 g of the culture solution was placed in a homogenizer cup, 200 ml of distilled water was added, homogenized at 20,000 rpm for 2 minutes, and heat-treated at 95 ° C. for 6 hours. 800 mL of 99% ethanol was added and stirred at 4 ° C. for 24 h. The supernatant was collected by centrifugation at 6,000 rpm for 30 minutes, filtered using a filter paper (Whatman No. 2), and concentrated under reduced pressure at 60 ° C. It was dissolved again by adding water, filtered through a filter of 0.45 mu m, and the solution was adjusted to 1 ml and analyzed by HPLC. Cordycepin standard for comparison was C3394 from Sigma-Aldrich Chemical. Column is μbondapak C18 (300mm LX 3.9mm ID, Waters), Detecter is UV detector (260nm, Waters), Mobile phase is 0.1% TFA: Acetonitrile (9: 1) , Flow rate was 0.5 ml / min.
1 is a result of pretreatment and dilution of 100 times the ovulation cultured Cordyceps sinensis for 20 days, according to the results. According to this result, about 17.8mg of cordycepin per egg of Cordyceps sinensis, 356mg based on the culture / l of cordycepin was produced.
In addition, a comparison of cordycepin production data performed in various organs is shown in Table 1 below.
(Applicant)
356 mg / l
Claims (4)
b) inoculating the pre-culture solution to the sterilized ovulation obtained in step a) and incubating with aeration of 0.1 ~ 0.5vvm; cordycepin production method using undigested ovulation comprising a.
b) purifying cordycepin after the step; cordycepin production method characterized in that the addition.
b) step of producing cordycepin, characterized in that performed for 5 to 30 days.
Cordyceps is cordyceps millitaris, characterized in that the cordycepin production method.
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| Application Number | Priority Date | Filing Date | Title |
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| KR1020100139750A KR20120077691A (en) | 2010-12-31 | 2010-12-31 | A cordycepin producing method using unhatched egg |
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| KR1020100139750A KR20120077691A (en) | 2010-12-31 | 2010-12-31 | A cordycepin producing method using unhatched egg |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103563650A (en) * | 2013-10-17 | 2014-02-12 | 烟台金刚生源生物科技有限公司 | Technology for culturing bird-sourced cordyceps |
| CN104327139A (en) * | 2014-09-28 | 2015-02-04 | 西北农林科技大学 | Preparation method of cordycepin crystal |
| CN110295117A (en) * | 2019-07-26 | 2019-10-01 | 周口职业技术学院 | The culture and preparation method of a kind of Cordceps militaris in egg |
-
2010
- 2010-12-31 KR KR1020100139750A patent/KR20120077691A/en not_active Application Discontinuation
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103563650A (en) * | 2013-10-17 | 2014-02-12 | 烟台金刚生源生物科技有限公司 | Technology for culturing bird-sourced cordyceps |
| CN103563650B (en) * | 2013-10-17 | 2015-04-22 | 烟台金刚生源生物科技有限公司 | Technology for culturing bird-sourced cordyceps |
| CN104327139A (en) * | 2014-09-28 | 2015-02-04 | 西北农林科技大学 | Preparation method of cordycepin crystal |
| CN110295117A (en) * | 2019-07-26 | 2019-10-01 | 周口职业技术学院 | The culture and preparation method of a kind of Cordceps militaris in egg |
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